Project Summary The regulatory kinase Stk1 controls genes involved in antibiotic resistance, biofilm formation, and toxin expression in virulent strains of Staphylococcus aureus. Inhibition of Stk1 in vitro potentiates methicillin-resistant S. aureus (MRSA) to b-lactam antibiotics and inhibits biofilm formation, making it an attractive target for the development of novel antibiotic adjuvant and antibiofilm therapies. However, studies of stk1 deletion mutants in vivo have many raised questions about the role of Stk1 in infection. In some MRSA strains and infection models, deletion of stk1 attenuates infection, whereas in other models and strains the stk1 deletion mutant displays enhanced virulence. Because only a few substrates of Stk1 have been identified, it is difficult to predict the full consequences of Stk1 inhibition. Additionally, strain-specific differences in the downstream genes whose expression is affected by Stk1 phosphorylation has prevented comprehensive understanding of Stk1-mediated gene expression in S. aureus. Our long-term goal is to develop novel anti-virulence treatments to combat persistent and antibiotic resistant bacterial infections. Our overall objectives in this proposal are to develop Stk1 inhibitors as chemical probes that can identify strain-specific differences in Stk1 inhibition and expand our knowledge of Stk1-mediated virulence gene expression in medically-relevant strains of S. aureus. Specifically, we will use our designed chemical probes to interrogate the inhibitor binding site of Stk1 across several strains of S. aureus to elucidate strain-specific differences that could affect development of broadly active inhibitors. Lead probes will then be used to identify novel antibiotic classes whose susceptibility is affected by Stk1 activity. Finally, we will use a combined transcriptomic and proteomic approach to identify novel Stk1 substrates and map downstream genes whose expression is affected by Stk1. Together, this will enhance existing knowledge of virulence pathways and their regulatory mechanisms in S. aureus. These results will have a significant impact as they will expand our understanding of gene regulation in S. aureus and provide necessary information for evaluation of Stk1 as a therapeutic target, thereby providing crucial new information for the development of novel antibacterial therapies.